PROJECT SUMMARY ABSTRACT Public Health Problem In the U.S., 1,144,500 people aged 13 years and older are living with HIV infection, with approximately 180,900 (15.8%) others infected but undiagnosed. For most HIV-positive individuals, available treatments can only control HIV infection and delay progression to AIDS. HAART has substantial side effects and fails to prevent at least 50% of patients from developing HIV-1 associated neurocognitive disorders. Pre-exposure prophylaxis (PrEP) has been an exciting development for reduction of HIV acquisition risk and transmission, but it remains unclear when it is safe to discontinue PrEP treatment. The ultimate value of the SBIR Phase II project will demonstrate the ability of our technology to predict time to viral load rebound after treatment interruption, which remains a critical unmet need in the HIV cure field. Issues with Current Solutions & How Product Meets Unmet Needs Current methods of quantifying the latent reserve include the quantitative viral outgrowth assay (Q-VOA), PCR and RT-PCR. Q-VOA is a time and resource intensive procedure while RT-PCR can be used to detect viral RNA to 20-50 virus particles per mL and thus reduces the time to result of QVOA and is generally applicable for measuring viral load, but does not directly detect replication-competent latent HIV-infected cells. Jan Biotech?s HIV qLDR offers clear advantages including: 1) detection of latent reservoir cells in the blood without activation; 2) direct detection of RNA to improve quantitative ability that is lost in other assays; 3) nonenzymatic amplification, which relieves the need for RNA purification and its attendant loss of RNA; 4) detection of short segments of RNA, providing effective detection even in the presence of RNase degradative enzymes; 5) Jan Biotech?s qLDR PNA probes enable the use of short sequences complementary to highly conserved regions of the HIV-1 genome, which allows for breadth of detection across the highly genetically diverse HIV-1 subtypes. Summary of Approach The Phase I qLDR probe sets differentiated between latent and activated HIV-infected cells in peripheral blood, which has great promise for accurate measurement of the level of the replication-competent latent reservoir. The Phase II Specific Aims will demonstrate the ability of the assay to predict time to viral load rebound after treatment interruption using ex vivo and in vivo samples. Testing will evaluate the utility of the assay as a clinical endpoint to guide treatment interruption decisions and facilitate HIV remission and cure discovery. Collaborators and Unique Resources Jan Biotech, Inc., with expertise in molecular diagnostic development, will consult with Jonathan Li, MD, MMSc Assistant Professor of Medicine, Harvard Medical School, in a AIDS Clinical Trials Group (ACTG) study. Judith Currier, MD, MSc, Vice Chair of the ACTG Network will oversee sample collection from the treatment interruption study, A5345; Q-VOA cross-validation testing will be performed by Southern Research Institute. Phase II Specific Aims Specific Aim 1: HIV qLDR prediction of replication-competent latent reservoir Task 1: Optimization of qLDR probe sets Task 2: Ex vivo rebound after treatment interruption (TI) Specific Aim 2: HIV qLDR prediction of HIV rebound during Analytical Treatment Interruption Task 1: HIV qLDR prediction of Viral Rebound after Analytical Treatment Interruption (ATI) Task 2: Comparison of HIV qLDR to other assays used in ATI study Specific Aim 3: Preclinical Validation of HIV qLDR Task 1: Validation of qLDR performance characteristics Task 2: QVOA validation Market after Phase II Completion Both Gilead and Bionor are commercially interested in the technology and look forward to results generated from the Phase II and the ACTG study. Regulatory approval will be sought for commercialization.